This proposal outlines studies to investigate several working hypotheses based upon recent observations from this laboratory and by others which pertain to cellular mechanisms of canalicular bile formation. The proposed studies will (1) examine the possibility that certain bile acids as well as bile acid-independent choleretics stimulate bile flow at the level of the canaliculus by stimulating extrusion of protons across the hepatocyte plasma membrane and raising intracellular pH (pHi) and HCO3- concentratiion, (2) explore the role that Na+/H+ exchange, enhanced ATP-dependent H+ transport mediated by vesicular insertion of H+-ATPase pump units into the plasma membrane, or other possible H+ transport mechanisms play in the postulated dynamic relationship between regulation of pHi and HCO3- secretion, (3) determine whether hepatocytes actively accumulate Cl- via Na+/K+/Cl- cotransport or HCO3-/Cl- exchange and examine the possibility that active Cl- transport plays a role in bile formation, (4) determine whether the Na+-coupled uptake of bile acids is electrogenic or electroneutral, and further explore the mechanisms by which hepatocytes preserve the electrochemical Na+ gradient during the Na+-coupled uptake of bile acids and/or amino acids, (5) quantitatively characterize the relationship between the vesicular blood-to-bile transport of fluid phase markers and overall fluid phase endocytosis by hepatocytes, and (6) explore the level in the endocytic pathway at which ligands destined for bile are segragated from those bound for lysosomes as well as the role of vesicle acidification in this segregation process. These hypotheses will be examined using conventional models and techniques (intact perfused liver, isolated or cultured cells, plasma membrane vesicles) as well as techniques recently acquired or developed by the investigators (conventional and ion-selective microelectrodes, fluorimetric analysis of intact cells and individual vesicles) which have had little prior application to liver and can directly address the novel questions posed. The studies are expected to provide new insight into cellular mechanisms of canalicular bile formation as well as into fundamental, yet poorly understood aspects of epithelial transport in general. The findings will thus be of importance to our current understanding of the pathophysiology of intrahepatic cholestasis as well as of other idseases involving abnormalities in transport, including those affecting the kidney and intestine, cystic fibrosis, and hypertension.